Hypoxia of human solid tumors is associated with reduced cure rates with radiotherapy and with increased distant metastases. However, hypoxia also represents an attractive therapeutic target, since drugs can be designed that are activated to become cytotoxic only in the hypoxic environment of solid tumors. Tirapazamine hypoxic cells in solid tumors. Further, it exhibits a strong synergistic cell kill with the widely used anti- cancer agents cisplatin and carboplatin. Both of these interactions are being exploited in clinical studies. However, the mechanisms for the selective hypoxic cytotoxicity and for the synergistic interaction with cisplatin are only poorly understood. In particular, the mechanism by which TPZ produces DNA double-strand breaks (dsb's), the identify of the enzyme(s) responsible for the metabolism of TPZ that leads to cell killing, as well as the mechanism for the TPZ-cisplatin interaction, are all unknown. The recent construction of a comprehensive set of mutants of S. cerevisiae with homozygous deletions of each open reading frame (ORF) has provided a powerful new tool to investigate these mechanisms. In addition, and complementary to use of the deletion mutant pool, gene expression profiling also allows information to be obtained on the mechanism of action of drugs. We propose to use both of these technologies with TPZ and other anti-cancer agents of known mechanism of action to determine the molecular mechanism in yeast for the action of TPZ under hypoxia, for the identity of metabolizing enzymes and for the mechanism of the synergy with cisplatin. Identification of yeast genes will allow us to identify and study the human homologs. Finally, we also intend to use the genetic suppressor element (GSE) technology to identify directly in human cells the enzyme(s) involved in TPZ metabolism leading to hypoxic cell kill. Identification of these mechanisms and of the enzyme(s) responsible for cell killing by TPZ will aid both in selection of patients or tumor types for clinical use of TPZ and in the development of second generation agents based on TPZ.
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